Gene Expression Profiling in Slow-Type Calf Soleus Muscle of 30 Days Space-Flown Mice

Abstract

Microgravity exposure as well as chronic disuse are two main causes of skeletal muscle atrophy in animals and humans. The antigravity calf soleus is a reference postural muscle to investigate the mechanism of disuse-induced maladaptation and plasticity of human and rodent (rats or mice) skeletal musculature. Here, we report microgravity-induced global gene expression changes in space-flown mouse skeletal muscle and the identification of yet unknown disuse susceptible transcripts found in soleus (a mainly slow phenotype) but not in extensor digitorum longus (a mainly fast phenotype dorsiflexor as functional counterpart to soleus). Adult C57Bl/N6 male mice (n = 5) flew aboard a biosatellite for 30 days on orbit (BION-M1 mission, 2013), a sex and age-matched cohort were housed in standard vivarium cages (n = 5), or in a replicate flight habitat as ground control (n = 5). Next to disuse atrophy signs (reduced size and myofiber phenotype I to II type shift) as much as 680 differentially expressed genes were found in the space-flown soleus, and only 72 in extensor digitorum longus (only 24 genes in common) compared to ground controls. Altered expression of gene transcripts matched key biological processes (contractile machinery, calcium homeostasis, muscle development, cell metabolism, inflammatory and oxidative stress response). Some transcripts (Fzd9, Casq2, Kcnma1, Ppara, Myf6) were further validated by quantitative real-time PCR (qRT-PCR). Besides previous reports on other leg muscle types we put forth for the first time a complete set of microgravity susceptible gene transcripts in soleus of mice as promising new biomarkers or targets for optimization of physical countermeasures and rehabilitation protocols to overcome disuse atrophy conditions in different clinical settings, rehabilitation and spaceflight.

Fig 1. Morphological analysis of mouse soleus and EDL skeletal muscle flown on board the BION-M1 biosatellite for 30 days.

Upper panel, insets show light microscopy images of Haematoxylin Eosin merged to immunofluorescence images of soleus (SOL) and EDL stained for MyHC isoforms (MyHC I: blue, IIa: green, IIb: red) in flown (BF) and control (BG) mice. Lower panel, scatter plots showing the quantification of the myofiber cross sectional area (CSA) and type composition in soleus (n = 2) and EDL (n = 2). Scale bar: 100 μm.

Fig 2. Principal Component Analysis (PCA) of gene expression data in soleus and EDL.

PCA analysis of gene expression data in soleus (A) and EDL (B) highlights the high sensitivity of soleus to microgravity exposure compared to EDL.

Fig 3. Venn diagrams and heat maps showing genes differentially regulated in soleus and EDL of BION-M1 space flown mice.

A, B, Venn diagrams showing the number of genes differentially regulated comparing space flown (BF) with ground controls (BG and FC) in soleus (A) and EDL (B). Comparisons between the three different experimental groups are presented (BF vs. BG, FC vs. BG and BF vs. FC). C, D, hierarchical clustering centred on BF vs. BG in soleus (C, ordered BG-FC-BF) and EDL (D, ordered BF-FC-BG). The differentially regulated genes meeting FDR < 0,05 (soleus), p < 0.05 (EDL) and < -2 & > +2 fold change criteria are included in the heat maps.

Fig 4. Biological functions regulated in soleus of BION-M1 flown mice.

Pie graph showing the percentage of genes linked to biological functions differentially expressed in soleus in BF group compared to ground control (BG) group. GO enrichment tool of Partek^® Genomics Suite^® software was used to perform the analysis. The Fisher's Exact test on the counts of genes was used to identify key functional groups in biological functions with respect to an enrichment p value < 0.05 and more than 8 genes per group.

Fig 5. Common genes differentially regulated in soleus and EDL in BION-M1 flown mice.

A, Venn diagram showing the number of genes differentially regulated in soleus and EDL following 30 days of microgravity exposure (BF vs. BG). B, C, D, bar charts showing the fold change in the expression of cyclin-dependent kinase inhibitor 1A (P21) (B), the myogenic factor 6 (C) and synaptojanin 2 (D) in both soleus and EDL muscles of flown mice (BF) compared to ground controls (BG and FC). The table includes the fold change and p-values for each gene depicted as bar graph in B-D.

Fig 6. Real time qPCR analysis of selected genes differentially regulated in soleus of BF vs. BG mice.

Expression levels of frizzled homolog 9 (Fzd9), calsequestrin 2 (Casq2), potassium large conductance calcium-activated channel, subfamily M, alpha member 1 (Kcnma1), peroxisome proliferator activated receptor alpha (Ppara), actinin alpha 3 (Actn3), myogenic factor 6 (Myf6), myostatin (Mstn) and Muscle RING Finger 1 (MuRF1) were evaluated by real-time quantitative PCR in soleus of flown (BF) and ground control mice (BG). Actb (beta actin) was used as housekeeping to calculate the delta Ct of the selected genes. Graph shows ΔC_t ± SEM; *** p < 0,0007, ** p < 0,006 and * p < 0,03.